Sandbox 32

=Introduction= Trypsin is a medium size globular protein that functions as a pancreatic serine protease. This enzyme hydrolyzes bonds by cleaving peptides on the C-terminal side of the amino acid residues lysine and arginine. It has also been shown that cleavage will not occur if there is a proline residue on the carboxyl side of the cleavage site. Trypsin was first discovered in 1876 by Kuhne, who investigated the proteolytic activity of the enzyme. In 1931 the enzyme was purified by crystallization by Norothrop and Kunitz and later in 1974 the three dimensional structure of trypsin was determined. Throughout the 1990's the role of trypsin in hereditary pancreatitis and the mutation that causes it was discovered. Today trypsin is used in the development of cell and tissue protocols, as well as in the medical field to determine the role of trypsin in pancreatic diseases.

Trypsinogen is the precursor form or zymogen of Trypsin. Zymogens are enzyme precursors that are made and secreted in the lysosome of the cell. Zymogens are not active until they go through a chemical process such as hydrolysis, cleavage, or other cleavages that reveal the active site. The zymogen precursor is necessary in order to prevent the destruction of cellular proteins and to allow the enzyme to be in it's active state only when in appropriate conditions. Trypsinogen is specifically produced in the exocrine cells of the pancreas. There are three isoforms of Trypsinogen that are secreted by the pancreas. The precursor is only activated when it reaches the lumen of the small intestine. This activation occurs through the help of an enteropeptidase and once activated trypsin stimulates the formation of more trypsinogen. The structure of Bovine Trypsinogen is shown in the figure to the right.

Trypsin has many applications due to fact that it is easily purified in high quantities. The trypsin enzyme is often used in the research setting to digest proteins and then identify the resulting peptides using mass spectrometry. Trypsin has many uses in the medical field such as dissolving blood clots and treating inflammation. Other applications include its use in pre-digesting of baby food, fingerprinting and sequencing work, and environmental monitoring.

=Structure= The pathway of the protein can be followed from N-terminus of the protein (blue) to the C-terminus of the protein (red). Trypsin has many important structural aspects. The secondary structures are shown this figure (Secondary Structure). The main backbone of the trypsin protein is shown in yellow (main backbone). Trypsin has two alpha helices shown in blue (alpha helices) and two beta sheets shown in green (beta Sheets). The beta sheets in the Trypsin protein are antiparallel to each other and connected by a Beta-hairpin turn.

This image shows the polarity of the residues in the protein. The polar areas of the protein are shown in pink, while the non-polar areas of the molecule are shown in light blue. The polarity of the individual amino acid residues can be seen better in the stick model or the spacefill model. The polar amino acid residues are again shown in pink, while the non-polar amino acid residues are shown in blue. By rotating the three representations of the polar versus non-polar areas of the protein to an aerial view, it can be seen that the polar (hydrophilic) areas are located toward the outside of the protein, while the non-polar (hydrophobic) areas are located toward the inside of the protein.

The charge of the different components of Trypsin are shown in this figure (charge figure). The cationic (+) atoms are shown in blue, while the anionic (-) are shown in red. These charged aspects of the protein face the outside environment surrounding the protein. The light purple parts of the protein are uncharged and the gray portions of the protein are hydrophobic. These portions of the protein make up the hydrophobic core of the protein. The charged and uncharged portions of the protein directly relate to the hydrophilic and hydrophobic character of the protein.

The side chains are attached to the amino acids residues that make up the protein. In this figure the side chains of the protein are shown in pink and the rest of the protein is shown in grey. There are numerous different side chains represented on the protein. Some of the side chains are aromatic, while others are not. Some side chains are charged, while others are uncharged. The type of side chain it is and therefore the corresponding amino acid can be determined by the charge on the side chain.

There are three disulfide bonds in the Trypsin protein. These bonds occur between Cysteine residues and are shown in yellow in this image of the protein. The remainder of the protein is shown in grey. Disulfide bonds in a protein act as stabilizing forces that occur within and between polypeptide chains.

Ions and Their Intermolecular Forces Between them and the Protein
The Trypsin protein has ligands that interact with other aspects of the protein. These ligands are shown in the figure as the red and yellow compounds, while the remainder of the protein is white in color. Three of the four ligands contain four oxygen atoms (red) and one Sulfur atom (yellow). The final ligand contains two Oxygen atoms and one Sulfur atom. 

The interactions between the Sulfur atoms are shown in this figure. The Sulfur atoms are shown in green and the ball and stick model shows the parts of the protein that are interacting with the corresponding Sulfur atoms. The remaining parts of the protein interacting with the green Sulfur atoms are the certain parts of the nearby amino acid residues. The Sulfur atoms shown in white in this figure can be ignored since they are related to the Sulfur atoms in the disulfide bonds.

The interactions between the <scene name='Sandbox_32/Oxygen_ion_interactions/2'>Oxygen atoms are shown in this figure. The Oxygen atoms are shown in red and the ball and stick model shows the parts of the protein that are interacting with the corresponding Oxygen atoms.

The composition of the Trypsin protein can be seen when surrounded <scene name='Sandbox_32/Compostion/2'>water molecules. This image shows where and how water molecules bond to the protein. The water molecules are blue, while the protein is an off-white color. These interactions are important when it comes to how the protein interacts with its environment.

=Catalytic Mechanism= The function of Trypsin is to break down peptides using a hydrolysis reaction into amino acid building blocks. This mechanism is a general catalytic mechanism that all Serine proteases use. The active site where this mechanism occurs in Trypsin is composed of three amino acids and called a <scene name='Sandbox_45/Ctriadd102h57s195/4'>catalytic triad. The three catalytic residues are Serine 195, Histidine 57, and Aspartate 102. The structure of the catalytic triad and the mechanism are shown in the figures to the right. In the mechanism, serine is bonded to the imidazole ring of the histidine. When histidine accepts a proton from serine an alkoxide nucleophile is formed. This nucleophile attacks the substrate when the substrate is present. The role of the aspartate residue is hold histidine in the proper position to make it a good proton acceptor. What makes this mechanism works is that a pocket if formed from the three residues and the three residues function to hold each other in proper position for nucleophilic attack. The steps of the mechanism involve two tetrahedral intermediates and an Acyl-enzyme intermediate. The mechanism can be followed in more detail in the figure on the right.

Oxyanion Hole
An important motif that is formed in this reaction is an oxyanion hole. This is also shown in the figure to the right. This oxyanion hole is specifically formed between the amide hydrogen atoms of Serine 195 and Glycine 193. This oxyanion hole stabilizes the tetrahedral intermediate through the distribution of negative charge to the cleaved amide.

=Comparison to Chymotrypsin and Elastase= <applet scene='Sandbox_32/Chymotrypsin/1' size='275' frame='true' align='true' align='left' caption='Structure of Chymotrypsin and Elastase.'/> Trypsin, chymotrypsin, and elastase are all digestive enzymes that are produced in the pancreas and catalyze the hydrolysis of peptide bonds. Each of these enzymes has different specificities in regards to the side chains next to the peptide bond. Chymotrypsin prefers a large hydrophobic residue, trypsin is specific for a positively charged residue, and elastase prefers a small neutral residue. Chymotrypsin, trypsin and elastase are all proteins that contain a catalytic mechanism and hydrolyze peptides using the serine protease mechanism. Chymotrypsin and elastase are both homologs of Trypsin since they are 40% alike in structure and composition. In the <scene name='Sandbox_32/Chymotrypsin/2'>Chymotrypsin structure shown the alpha helices are blue, the beta sheets are green, and the remainder of the protein is red. In the <scene name='Sandbox_32/Elastase/2'>Elastase structure shown the alpha helices are in red, the beta sheets are yellow, and the remainder of the protein is orange.